Naureen Javeed

Adrenomedullin is Up-regulated in Patients With Pancreatic Cancer and Causes Insulin Resistance in β Cells and Mice

BACKGROUND & AIMS New-onset diabetes in patients with pancreatic cancer is likely to be a paraneoplastic phenomenon caused by tumor-secreted products. We aimed to identify the diabetogenic secretory product(s) of pancreatic cancer. METHODS Using microarray analysis, we identified adrenomedullin as a potential mediator of diabetes in patients with pancreatic cancer. Adrenomedullin was up-regulated in pancreatic cancer cell lines, in which supernatants reduced insulin signaling in beta cell lines. We performed quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry on human pancreatic cancer and healthy pancreatic tissues (controls) to determine expression of adrenomedullin messenger RNA and protein, respectively. We studied the effects of adrenomedullin on insulin secretion by beta cell lines and whole islets from mice and on glucose tolerance in pancreatic xenografts in mice. We measured plasma levels of adrenomedullin in patients with pancreatic cancer, patients with type 2 diabetes mellitus, and individuals with normal fasting glucose levels (controls). RESULTS Levels of adrenomedullin messenger RNA and protein were increased in human pancreatic cancer samples compared with controls. Adrenomedullin and conditioned media from pancreatic cell lines inhibited glucose-stimulated insulin secretion from beta cell lines and islets isolated from mice; the effects of conditioned media from pancreatic cancer cells were reduced by small hairpin RNA-mediated knockdown of adrenomedullin. Conversely, overexpression of adrenomedullin in mice with pancreatic cancer led to glucose intolerance. Mean plasma levels of adrenomedullin (femtomoles per liter) were higher in patients with pancreatic cancer compared with patients with diabetes or controls. Levels of adrenomedullin were higher in patients with pancreatic cancer who developed diabetes compared those who did not. CONCLUSIONS Adrenomedullin is up-regulated in patients with pancreatic cancer and causes insulin resistance in β cells and mice.

Pathogenesis of pancreatic cancer exosome-induced lipolysis in adipose tissue

Background and objectives New-onset diabetes and concomitant weight loss occurring several months before the clinical presentation of pancreatic cancer (PC) appear to be paraneoplastic phenomena caused by tumour-secreted products. Our recent findings have shown exosomal adrenomedullin (AM) is important in development of diabetes in PC. Adipose tissue lipolysis might explain early onset weight loss in PC. We hypothesise that lipolysis-inducing cargo is carried in exosomes shed by PC and is responsible for the paraneoplastic effects. Therefore, in this study we investigate if exosomes secreted by PC induce lipolysis in adipocytes and explore the role of AM in PC-exosomes as the mediator of this lipolysis. Design Exosomes from patient-derived cell lines and from plasma of patients with PC and non-PC controls were isolated and characterised. Differentiated murine (3T3-L1) and human adipocytes were exposed to these exosomes to study lipolysis. Glycerol assay and western blotting were used to study lipolysis. Duolink Assay was used to study AM and adrenomedullin receptor (ADMR) interaction in adipocytes treated with exosomes. Results In murine and human adipocytes, we found that both AM and PC-exosomes promoted lipolysis, which was abrogated by ADMR blockade. AM interacted with its receptor on the adipocytes, activated p38 and extracellular signal-regulated (ERK1/2) mitogen-activated protein kinases and promoted lipolysis by phosphorylating hormone-sensitive lipase. PKH67-labelled PC-exosomes were readily internalised into adipocytes and involved both caveolin and macropinocytosis as possible mechanisms for endocytosis. Conclusions PC-secreted exosomes induce lipolysis in subcutaneous adipose tissue; exosomal AM is a candidate mediator of this effect.

Pancreatic cancer-derived exosomes cause paraneoplastic β-cell dysfunction

Purpose: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer–derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion. Experimental Methods: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown. Results: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species. Conclusions: Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin+/CA19-9+ exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response. Clin Cancer Res; 21(7); 1722–33. ©2014 AACR. See related commentary by Korc, p. 1508

Exosomes and their role in the micro-/macro-environment: a comprehensive review

The importance of extracellular vesicles (EVs) in cell-cell communication has long been recognized due to their ability to transfer important cellular cargoes such as DNA, mRNA, miRNAs, and proteins to target cells. Compelling evidence supports the role of EVs in the horizontal transfer of cellular material which has the potential to influence normal cellular physiology and promote various disease states. Of the different types of EVs, exosomes have garnered much attention in the past decade due to their abundance in various biological fluids and ability to affect multiple organ systems. The main focus of this review will be on cancer and how cancer-derived exosomes are important mediators of metastasis, angiogenesis, immune modulation, and the tumor macro-/microenvironment. We will also discuss exosomes as potential biomarkers for cancers due to their abundance in biological fluids, ease of uptake, and cellular content. Exosome use in diagnosis, prognosis, and in establishing treatment regimens has enormous potential to revolutionize patient care.

Dopamine D2 receptor agonists inhibit lung cancer progression by reducing angiogenesis and tumor infiltrating myeloid derived suppressor cells

We sought to determine whether Dopamine D2 Receptor (D2R) agonists inhibit lung tumor progression and identify subpopulations of lung cancer patients that benefit most from D2R agonist therapy. We demonstrate D2R agonists abrogate lung tumor progression in syngeneic (LLC1) and human xenograft (A549) orthotopic murine models through inhibition of tumor angiogenesis and reduction of tumor infiltrating myeloid derived suppressor cells. Pathological examination of human lung cancer tissue revealed a positive correlation between endothelial D2R expression and tumor stage. Lung cancer patients with a smoking history exhibited greater levels of D2R in lung endothelium. Our results suggest D2R agonists may represent a promising individualized therapy for lung cancer patients with high levels of endothelial D2R expression and a smoking history.

Immunosuppressive CD14+HLA-DRlo/neg monocytes are elevated in pancreatic cancer and “primed” by tumor-derived exosomes

ABSTRACT Immunological strategies to treat pancreatic cancer offer new therapeutic approaches to improve patient outcomes. Understanding alterations in the immune systems of pancreatic cancer patients will likely lead to advances in immunotherapy for the disease. We profiled peripheral blood leukocytes from pancreatic cancer patients (n = 22) and age-matched controls (n = 20) using flow cytometry. Immune profiling of pancreatic cancer patients identified phenotypic changes in various immune cell populations, including a population of immunosuppressive monocytes (CD14+HLA-DRlo/neg), which were shown to be increased in these patients. There was a correlation between the levels of CD14+ monocytes and the levels of CD14+HLA-DRlo/neg monocytes in peripheral blood from pancreatic cancer patients. HLA-DR downregulation of monocytes was shown to occur through pancreatic cancer-derived exosome interactions with monocytes. In an in vitro model, exosomes from patient-derived xenograft cell lines and patient plasma decreased HLA-DR expression on CD14+ monocytes. Additionally, tumor-derived exosomes caused immune suppression in monocytes through altered STAT3 signaling, induction of arginase expression, and reactive oxygen species. These findings provide novel insights into the mechanisms that govern immunosuppression in pancreatic cancer. Understanding monocyte–exosome interactions could lead to novel immunotherapies for this disease.

Circadian Etiology of Type 2 Diabetes Mellitus

The epidemic of Type 2 diabetes mellitus necessitates development of novel therapeutic and preventative strategies to attenuate expansion of this debilitating disease. Evidence links the circadian system to various aspects of diabetes pathophysiology and treatment. The aim of this review will be to outline the rationale for therapeutic targeting of the circadian system in the treatment and prevention of Type 2 diabetes mellitus and consequent metabolic comorbidities.

Inhibition of TBK1/IKKε Promotes Regeneration of Pancreatic β-cells

Abstractβ-cell proliferation induction is a promising therapeutic strategy to restore β-cell mass. By screening small molecules in a transgenic zebrafish model of type 1 diabetes, we identified inhibitors of non-canonical IκB kinases (IKKs), TANK-binding kinase 1 (TBK1) and IκB kinase ε (IKKε), as enhancers of β-cell regeneration. The most potent β-cell regeneration enhancer was a cinnamic acid derivative (E)-3-(3-phenylbenzo[c]isoxazol-5-yl)acrylic acid (PIAA), which, acting through the cAMP-dependent protein kinase A (PKA), stimulated β-cell-specific proliferation by increasing cyclic AMP (cAMP) levels and mechanistic target of rapamycin (mTOR) activity. A combination of PIAA and cilostamide, an inhibitor of β-cell-enriched cAMP hydrolyzing enzyme phosphodiesterase (PDE) 3, enhanced β-cell proliferation, whereas overexpression of PDE3 blunted the mitogenic effect of PIAA in zebrafish. PIAA augmented proliferation of INS-1β-cells and β-cells in mammalian islets including human islets with elevation in cAMP levels and insulin secretion. PIAA improved glycemic control in streptozotocin (STZ)-induced diabetic mice with increases in β-cell proliferation, β-cell area, and insulin content in the pancreas. Collectively, these data reveal an evolutionarily conserved and critical role of TBK1/IKKε suppression in expanding functional β-cell mass.

Translational Potential of Tumor Exosomes in Diagnosis and Therapy

Abstract Exosomes are endocytic-derived nanovesicles that are secreted by nearly all cell types in the body. Not only do exosomes contain important bioactive cargoes, but they also serve as direct cellular conduits by transporting these cargoes both proximally and distally to target cells. The horizontal exchange of cellular material mediated by exosomes has been shown to influence both normal physiology and disease states, such as cancer. Compelling evidence supports the role of exosomes in altering the tumor microenvironment, in promoting angiogenesis and immune modulation, and in establishing the premetastatic niche. Therefore, exosomes may not only provide a potential mechanistic link to tumor metastasis but could also be used as cancer biomarkers for diagnostic and therapeutic regimes.

β cell self-renewal: Cyclin D2 to the rescue

Loss of functional b-cell mass is a key feature precipitating development of hyperglycemia in both Type 1 (T1DM) and Type 2 (T2DM) diabetes mellitus. In T1DM, b-cell loss occurs due to an autoimmune attack leading to near total ablation of b-cell mass whereas in T2DM, b-cell loss is more gradual (»50 to 65%) and manifests as a consequence of failure to expand b-cell mass in response to increased metabolic demand commonly attributed to obesity. Since b-cell failure is one of the key pathophysiological events precipitating the transition from normoglycemia to diabetes, therapeutic strategies aimed at restoration/regeneration of the endogenous b-cell mass have become a topic of concentrated investigation in diabetes research. Consequently, emerging b-cell regenerative approaches in diabetes include: de novo generation of stem cellderived b-cells, reprogramming/transdifferentiation of various endoderm-derived cell types to b-cells, and guided expansion of the endogenous b-cell mass through stimulation of b-cell self-renewal/replication. In this volume of Cell Cycle, Tschen, Zeng and colleagues reported that genetic induction of a key cell-cycle activating molecule, cyclin D2, was sufficient to drive postnatal b-cell replication, expand endogenous b-cell mass, and enhance b-cell regenerative potential in response to streptozotocin-induced injury. The major thrust of recent investigations into the regulation of b-cell replication has been on the molecular control of cellcycle progression with particular focus on the regulatory control of the G1/S checkpoint. Cellular progression through the G1/S checkpoint is regulated by complex molecular interactions between cell-cycle activators such as D, E and A cyclins, corresponding cyclin-dependent kinases (Cdks), and counterbalanced by a diverse set of cell cycle inhibitors (e.g. p16, p21, p27). It is important to emphasize that the molecular control of G1/S checkpoint molecules is the main downstream target of common b-cell mitogenic signaling pathways (e.g., Akt, PKC, cMyc, CREB, etc.). The aforementioned pathways are responsible for induction of b-cell proliferation during conditions associated with increased metabolic demand. Previous work from Georgia and Bhushan clearly demonstrated the unique requirement for cyclin D2 in postnatal b-cell replication and consequent establishment of adult b-cell mass and glucose tolerance. Follow up studies extended these observations to show that the expression of cyclin D2 is also essential for adult b-cell replication, and more importantly, compensatory expansion of functional b-cell mass in response to insulin resistance. These seminal observations established the role of cyclin D2 as an important G1/S regulatory protein controlling b-cell selfrenewal during critical periods of neonatal and adult b-cell expansion. Additionally, this work postulated the significance of cyclin D2 as a potential therapeutic target to promote b-cell expansion in diabetes. Although the aforementioned studies delivered compelling evidence for the role of cyclin D2 in b-cell self-renewal, two key questions remained unexplored. Firstly, the use of global cyclin D2 knockout mice left the possibility that effects of cyclin D2 deletion on b-cell mass were attributed to diminished de novo b-cell formation from putative pancreatic progenitors (i.e. b-cell neogenesis). Secondly, and perhaps most importantly, previous studies did not explore whether restoration or enhanced expression of cyclin D2 in b-cells is sufficient to restore/enhance b-cell functional mass in vivo. To address these questions, Tschen, Zeng et al. created a b-cell specific “knockin” mouse model overexpressing wildtype cyclin D2 using a Cre-mediated recombination system driven by the rat insulin 2 promoter. In a series of elegant studies, the authors reported that targeted b-cell expression of cyclin D2 resulted in increased b-cell self-renewal which coincided with doubling of postnatal b-cell mass, most notably, without compromising effects on b-cell functionality. This observation is particularly notable given recent reports suggesting repression of transcripts regulating glucose-stimulated insulin secretion in replicating b-cells. Moreover, with evidence to support the increased capacity of b-cells to undergo self-renewal through cyclin D2 overexpression, the authors also demonstrated enhanced regenerative capabilities of aged b-cells in response to streptozotocin-induced b-cell injury. This observation suggests that targeted expression of cyclin D2 may at least partially overcome age-dependent suppression of the b-cell replicative capacity. In summary, the ultimate goal of b-cell regenerative strategies would be to restore/replace functional b-cell mass that recapitulates cellular features of mature human islets. Future work will require translating these and other “rodent-based” findings to adult human b-cells, while demonstrating safety